Novel protease FMH-1

ABSTRACT

Polynucleotides encoding novel proteases designated “FMH-1” are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of copending application with Ser. No.08/675,123 filed on Jul. 3, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to protease proteins, nucleic acidsencoding such proteins, and methods of treatment using such proteins andpolynucleotides.

BACKGROUND OF THE INVENTION

[0003] Complex multicellular organisms employ numerous mechanisms toprotect and maintain the integrity of cellular components from whichthey are comprised. In the event of questionable or irreparable celldamage, one seemingly drastic measure available to a compromised cell isto commit suicide and to discretely remove itself for the altruisticbenefit of the organism as a whole. Cell suicide is triggered inresponse to pathogenic invasion (such as by a virus) to halt the spreadto neighboring cells, but it also occurs under nonthreatening conditionsto replace redundant or unnecessary cells, as occurs in tissuemorphogenesis and remodeling, and in the normal turnover of cells thatoccurs when they have exhausted their overall usefulness within theorganism. Nicholson, Nature Biotechnology 14:297 (1996).

[0004] The pathological manifestation of cell suicide, known as“apoptosis”, occurs as a result of a highly systematic and deliberatecell death pathway. These types of cell deaths have been appropriatelytermed “physiological cell death”, where in addition to this suicidepathway, death occurs in specific cells at a predetermined time.Apoptotic death is a highly ordered process that is characterized bynuclear changes such as chromatin condensation, fragmentation andmargination as well as interuucleosomal DNA cleavage (usually resultingin the hallmark DNA laddering), and by ultrastructural changes includingcytoskeletal disruption, cell shrinkage and membrane blebbing which thenleads to fragmentation of the dying cell into numerous membrane-boundapoptotic bodies that are subsequently engulfed by neighboring cells orprofessional macrophages in the final resolution of the suicide process.Apoptotic suicide has many advantages over other forms of cell death,owing principally to the membrane integrity that is maintainedthroughout the entire process. Necrotic cells, for example, leak theirconstituents into the surrounding extracellular space usually resultingin an inflammatory response. Nicholson, supra.

[0005] The recent explosion in interest in apoptosis is warranted giventhe substantial evidence that inappropriate apoptosis may contribute tothe pathology of several human diseases (including without limitationneurological diseases, such as Alzheimer's disease, amyotrophic lateralsclerosis, spinal muscular atrophy, neurological stroke damage,Parkinson's disease and Huntington's disease; immune system disorders,such as autoimmune syndromes, AIDS and type I diabetes; cardiovascularconditions, such as ischemic cardiac damage; proliferative conditions,such as solid tumors, lymphomas (such as follicular lymphoma) andleukemias; and others, such as pathogenic (including viral) infections,alopecia and aging. These can be divided into disorders of excessiveapoptosis (such as neurodegenerative disease or ischeric damage) andthose where insufficient apoptosis occurs (such as autoimmune syndromes,cancers and sustained pathogenic infections). Nicholson, supra.

[0006] In contrast to the above mentioned disorders, insufficientapoptosis is also associated with several human diseases. For example,many cancers are now believed to be the consequence of failed apoptoticcell death instead of enhanced cell growth as was originally thought.Two gene defects that are highly associated with proliferative disorders(p53 and Bcl-2) are now known to be regulators of the apoptotic process.Another example of flawed apoptosis is in autoimmune disorders where thefailure to remove autoreactive lymphocytes that arise duringdevelopment, or subsequent to an immune response, occurs. One form ofautoimmune disease, lupus erythematosus, may involve failure to completeapoptosis execution since most lupus autoantibodies recognize crypticepitopes within a distinct subset of polypeptides that are proteolyticcleavage victims in the cell death pathway. Finally, persistent viralinfections may be sustained and propagated because the normal host cellsuicide response that would be engaged following viral infection iscleverly suppressed by antiapoptotic viral gene products. Nicholson,supra.

[0007] Many of the clues which have implicated various components of thecell death pathway have arisen from disease association, geneticanalysis and in vitro reconstitution of apoptotic events. Collectively,this information has helped define a biochemical pathway that accountsfor many of the key events that occur in dying cells in vivo. Many partsof this pathway, particularly those involved in the effector events thatmediate the actual cell death process itself, appear to be common tomost cell types. At the heart of this process, proteases related tomammalian interleukin-1β converting enzyme (ICE) and to nematode CED-3appear to play an essential role. Several of the substrates that arecleaved by ICE/CED-3 like proteases at the onset of apoptosis have alsobeen identified. These include proteins that function in DNA repair aswell as structural proteins and regulatory enzymes. A fundamentalprinciple of apoptotic cell death thus appears to be the proteolyticdisabling of key homeostatic- and repair-processes as well as theobvious structural dismantling of the cell that is required tofacilitate its breakdown and subsequent packaging into apoptotic bodies.Moreover, ICE/CED-3 like proteolytic activities have been demonstratedto play a role in most if not all of these cleavage events. Nicholson,supra.

[0008] Molecular cloning has identified several human homologues of ICEand CED-3 including ICE_(rel)-II (TX, ICH-2), ICE_(rel)-III, ICH-1,CPP32 (apopain, Yama) Mch2 and Mch3 (ICE-LAP3) (Munday et al., J. Biol.Chem. 270:15870 (1995); Faucheu et al., EMBO J. 14:1914 (1995); Kamenset al., J. Biol. Chem. 270:15250 (1995); Kumar et al., Biochem. Biophys.Res. Commun. 185:1155 (1992); Kumar et al., Genes and Develop. 8:1613(1994); Wang et al.,Cell 78:739 (1994); Fernandes-Alnemri et al., J.Biol. Chem. 269:30761 (1994); Fernandes-Alnemri et al., Cancer Res.55:2737 (1995); Fernandes-Alnemri et al., Cancer Res. 55:6045 (1995);Duan et al., ICE-LAP3, a novel mammalian homolog of the Caenorhabditiselegans cell death protein CED-3 is activated during Fas- and tumornecrosis factor-induced apoptosis, J. Biol. Chem. in press (1996)).Nicholson, supra.

[0009] However, it would be desirable to identify other mammalian(including human) ICE/CED-3 like proteases.

SUMMARY OF THE INVENTION

[0010] In accordance with the present invention, polynucleotidesencoding a novel protease, FMH-1, and other related proteins aredisclosed. “FMH-1” is used throughout the present specification to referto both proteins and polynucleotides encoding those proteins and torefer to proteins and polynucleotides from all mammalian species.

[0011] In certain embodiments, the present invention provides for anisolated polynucleotide comprising a nucleotide sequence selected fromthe group consisting of:

[0012] (a) the nucleotide sequence of SEQ ID NO: 1 from nucleotide 121to nucleotide 1683;

[0013] (b) a nucleotide sequence capable of hybridizing to a nucleicacid sequence specified in (a);

[0014] (c) a nucleotide sequence encoding the amino acid sequence of SEQID NO: 2 and varying from the sequence of the nucleotide sequencespecified in (a) as a result of degeneracy of the genetic code;

[0015] (d) a nucleotide sequence comprising a fragment of (a) whichencodes an amino acid sequence comprising amino acids 399 to 403 of SEQID NO: 2;

[0016] (e) a nucleotide sequence comprising a fragment of (a) whichencodes an amino acid sequence comprising amino acids 20 to 95 of SEQ IDNO: 2; and

[0017] (f) an allelic variant of the nucleotide sequence specified in(a).

[0018] In preferred embodiments, the nucleotide sequence encodes for aprotein having an activity selected from the group consisting of FMH-1protease activity and adaptor binding activity. In other embodiments,the nucleotide sequence is operably linked to an expression controlsequence. Preferably, the polynucleotide encodes a human FMH-1 protein.Particularly preferred embodiments include polynucleotides comprisingthe nucleotide sequence of SEQ ID NO: 1 from nucleotide 121 tonucleotide 1683; a fragment of such sequence which encodes an amino acidsequence comprising amino acids 399 to 403 of SEQ ID NO: 2; and afragment of such sequence which encodes an amino acid sequencecomprising amino acids 20 to 95 of SEQ ID NO: 2.

[0019] The invention also provides for a host cell transformed with suchpolynucleotides, including mammalian cells. A process is also providedfor producing an FMH-1 protein, said process comprising: (a) growing aculture of the host cell of the invention in a suitable culture medium;and (b) purifying the FMH-1 protein from the culture. Proteins producedaccording to such processes are also provided.

[0020] Pharmaceutical compositions are also provided comprising suchpolynucleotides and a pharmaceutically acceptable carrier. In preferredembodiments, the polynucleotide is contained in a vector suitable forgene therapy and/or the composition further comprises agents capable ofincreasing the uptake of said polynucleotide by cells.

[0021] The invention further provides an isolated FMH-1 proteincomprising an amino acid sequence selected from the group consisting of:

[0022] (a) the amino acid sequence of SEQ ID NO: 2;

[0023] (b) fragments of (a) having FMH-1 protease activity; and

[0024] (c) fragments of (a) having adaptor binding activity.

[0025] Preferred embodiments include proteins comprising the amino acidsequence of SEQ ID NO: 2, proteins comprising amino acids 399 to 403 ofSEQ ID NO: 2; and proteins comprising amino acids 20 to 95 of SEQ ID NO:2. Pharmaceutical compositions comprising such proteins and apharmaceutically acceptable carrier are also provided.

[0026] The present invention also provides compositions comprising anantibody which specifically reacts with an FMH-1 protein of theinvention.

[0027] The invention also provides for methods of treating conditionsassociated with excessive or insufficient apoptosis by administering toa mammalian subject a therapeutically effective amount of apharmaceutical composition comprising an FMH-1 protein (as protein or inthe form of a polynucleotide, for example, through gene therapy).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0028] The inventors of the present application have identified andprovided a polynucleotide encoding a novel ICE/CED-3 like proteasedesignated “FMH-1”. SEQ ID NO: 1 provides the nucleotide sequence of acDNA encoding the human FMH-1 protein. SEQ ID NO: 2 provides the aminoacid sequence of the human FMH-1 protease.

[0029] Amino acids 331 to 472 of SEQ ID NO: 2 show substantialhomology/identity with other ICE/CED-3 like proteases and includes thedomain responsible for protease activity (including the QACQG motif atamino acids 399 to 403). Amino acids 20 to 95 of SEQ ID NO: 2 showsignificant homology to the adaptor protein binding domain of FADDprotein (Fas-associating protein with death domain) (amino acids 1-86 ofFADD) (see, Chinnaiyan et al., Cell 81:505 (1995)). It is believed thatthis domain in FMH-1 also constitutes an adaptor protein binding domain.

[0030] FMH-1 proteins comprising only one or several domains or portionsthereof may also be produced. Any forms of FMH-1 protein of less thanfull length are encompassed within the present invention and may beproduced by expressing a corresponding fragment of the polynucleotideencoding the FMH-1 protein (such as SEQ ID NO: 1). These correspondingpolynucleotide fragments are also part of the present invention.Modified polynucleotides as described above may be made by standardmolecular biology techniques, including site-directed mutagenesismethods which are known in the art or by the polymerase chain reactionusing appropriate oligonucleotide primers.

[0031] For the purposes of the present invention, a protein has “FMH-1protease activity” when it is capable of proteolysis in a mannercharacteristic of ICE/CED-3 like proteases. Protease activity of anFMH-1 protein can be measured by any of the assay methods reported inthe references cited above relating to cloning and characterization ofsuch proteases.

[0032] For the purposes of the present invention, a protein has “adaptorbinding activity” when it is capable of binding to adaptor proteins in amanner characteristic of ICE/CED-3 like proteases or FADD. Bindingactivity of an FMH-1 protein can be measured by any of the assay methodsreported in the references cited above relating to cloning andcharacterization of such proteases.

[0033] FMH-1 protein or fragments thereof having FMH-1 protease activitymay be fused to carrier molecules such as immunoglobulins. For example,an FMH-1 protein may be fused through “linker” sequences to the Fcportion of an immunoglobulin.

[0034] The invention also encompasses allelic variations of thenucleotide sequence as set forth in SEQ ID NO: 1, that is,naturally-occurring alternative forms of the isolated polynucleotide ofSEQ ID NO: 1 which also encode proteins having FMH-1 protease activity.Also included in the invention are isolated polynucleotides whichhybridize to the nucleotide sequence set forth in SEQ ID NO: 1 underhighly stringent (0.2×SSC at 65° C.), stringent (e.g. 4×SSC at 65° C. or50% formamide and 4×SSC at 42° C.), or relaxed (4×SSC at 50° C. or30-40% formamide at 42° C.) conditions. Isolated polynucleotides whichencode FMH-1 protein but which differ from the nucleotide sequence setforth in SEQ ID NO: 1 by virtue of the degeneracy of the genetic codeare also encompassed by the present invention. Variations in thenucleotide sequence as set forth in SEQ ID NO: 1 which are caused bypoint mutations or by induced modifications which enhance FMH-1 proteaseactivity, half-life or production level are also included in theinvention.

[0035] The isolated polynucleotides of the invention may be operablylinked to an expression control sequence such as the pMT2 or pEDexpression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19,4485-4490 (1991), in order to produce the FMH-1 protein recombinantly.Many suitable expression control sequences are known in the art. Generalmethods of expressing recombinant proteins are also known and areexemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). Asdefined herein “operably linked” means enzymatically or chemicallyligated to form a covalent bond between the isolated polynucleotide ofthe invention and the expression control sequence, in such a way thatthe FMH-1 protein is expressed by a host cell which has been transformed(transfected) with the ligated polynucleotide/expression controlsequence.

[0036] A number of types of cells may act as suitable host cells forexpression of the FMH-1 protein. Any cell type capable of expressingfunctional FMH-1 protein may be used. Suitable mammalian host cellsinclude, for example, monkey COS cells, Chinese Hamster Ovary (CHO)cells, human kidney 293 cells, human epidermal A43 1 cells, humanColo205 cells, 3T3 cells, CV-1 cells, other transformed primate celllines, normal diploid cells, cell strains derived from in vitro cultureof primary tissue, primary explants, HeLa cells, mouse L cells, BHK,HL-60, U937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12 or C2C12 cells.

[0037] The FMH-1 protein may also be produced by operably linking theisolated polynucleotide of the invention to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference. Soluble forms of the FMH-1 protein mayalso be produced in insect cells using appropriate isolatedpolynucleotides as described above.

[0038] Alternatively, the FMH-1 protein may be produced in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Suitableyeast strains include Saccharomyces cerevisiae, Schizosaccharomycespombe, Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous proteins. Suitable bacterial strains includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium, or anybacterial strain capable of expressing heterologous proteins.

[0039] The FMH-1 protein of the invention may also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide sequence encoding theFMH-1 protein.

[0040] The FMH-1 protein of the invention may be prepared by growing aculture transformed host cells under culture conditions necessary toexpress the desired protein. The resulting expressed protein may then bepurified from the culture medium or cell extracts. Soluble forms of theFMH-1 protein of the invention can be purified from conditioned media.Membrane-bound forms of FMH-1 protein of the invention can be purifiedby preparing a total membrane fraction from the expressing cell andextracting the membranes with a non-ionic detergent such as TritonX-100. The FMH-1 protein can be purified using methods known to thoseskilled in the art. For example, the FMH-1 protein of the invention canbe concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred (e.g., S-Sepharose® columns). The purification ofthe FMH-1 protein from culture supernatant may also include one or morecolumn steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; or by hydrophobicinteraction chromatography using such resins as phenyl ether, butylether, or propyl ether; or by immunoaffinity chromatography. Finally,one or more reverse-phase high performance liquid chromatography(RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gelhaving pendant methyl or other aliphatic groups, can be employed tofurther purify the FMH-1 protein. Some or all of the foregoingpurification steps, in various combinations or with other known methods,can also be employed to provide a substantially purified isolatedrecombinant protein.

[0041] FMH-1 proteins can also be purified by affinity chromatography onresins linked with peptides which are, or mimic, substratees for FMH-1,including without limitation targets of proteolysis and adatproproteins.

[0042] Preferably, the isolated FMH-1 protein is purified so that it issubstantially free of other mammalian proteins.

[0043] FMH-1 introduced by gene therapy may be used for inducingapoptosis in target cell populations. Fragments of the protein, againintroduced by gene therapy, may either induce or inhibit apoptosis intarget cell populations. FMH-1 may be used as a target for peptidemimetics or small molecule inhibitors which may induce or inhibitapoptosis depending on which region of the molecule they bind. Inductionor inhibition of apoptosis by the use of FMH-1, mimetics or inhibitorscan be used to treat conditions discussed above in which excessive orinsufficient apoptosis is implicated. The inhibition of apoptosis mayalso be used to enhance an immune reaction to weakly stimulatoryantigen.

[0044] Polynucleotides of the invention, including polynucleotidesencoding FMH-1 proteins, can be used in gene therapy in accordance withknown methods. The polynucleotide can be introduced as naked DNA, in aviral vector or other suitable vector known in the art. Suitable methodsinclude without limitation those described in WO95/26718, WO96/10038,WO93/19183, WO95/20660, U.S. Pat. No. 5,264,618, U.S. Pat. No.5,459,127, WO 90/11092, WO90/14074, WO91/16024, WO94/16716, WO89/01973and WO93/14778.

[0045] Isolated FMH-1 proteins, purified from cells or recombinantlyproduced, may be used as a pharmaceutical composition when combined witha pharmaceutically acceptable carrier. Such a composition may contain,in addition to FMH-1 protein and carrier, diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical compositions may also contain other additional activefactors or agents. Such additional factors and/or agents may be includedin the pharmaceutical composition to produce a synergistic effect withisolated FMH-1 protein, or to minimize side effects caused by theisolated FMH-1 protein.

[0046] The pharmaceutical composition of the invention may be in theform of a liposome in which isolated FMH-1 protein is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers which inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S.Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323, all of which are incorporated herein by reference.

[0047] As used herein, the term “therapeutically effective amount” meansthe total amount of each active component of the pharmaceuticalcomposition or method that is sufficient to show a meaningful patientbenefit, e.g., amelioration of symptoms of, healing of, or increase inrate of healing of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

[0048] Administration of isolated FMH-1 protein used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, or cutaneous, subcutaneous, or intravenousinjection. Intravenous administration to the patient is preferred.

[0049] When a therapeutically effective amount of isolated FMH-1 proteinis administered orally, isolated FMH-1 protein will be in the form of atablet, capsule, powder, solution or elixir. When administered in tabletform, the pharmaceutical composition of the invention may additionallycontain a solid carrier such as a gelatin or an adjuvant. The tablet,capsule, and powder contain from about 5 to 95% isolated FMH-1 protein,and preferably from about 25 to 90% isolated FMH-1 protein. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin such as peanut oil, mineral oil, soybeanoil, or sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of isolated FMH-1 protein, andpreferably from about 1 to 50% isolated FMH-1 protein.

[0050] When a therapeutically effective amount of isolated FMH-1 proteinis administered by intravenous, cutaneous or subcutaneous injection,isolated FMH-1 protein will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable protein solutions, having due regard to pH,isotonicity, stability, and the like, is within the skill in the art. Apreferred pharmaceutical composition for intravenous, cutaneous, orsubcutaneous injection should contain, in addition to isolated FMH-1protein an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additiveknown to those of skill in the art.

[0051] The amount of isolated FMH-1 protein in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of isolated FMH-1 protein with which totreat each individual patient. Initially, the attending physician willadminister low doses of isolated FMH-1 protein and observe the patient'sresponse. Larger doses of isolated FMH-1 protein may be administereduntil the optimal therapeutic effect is obtained for the patient, and atthat point the dosage is not generally increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.1 μgto about 100 mg of isolated FMH-1 protein per kg body weight.

[0052] The duration of intravenous therapy using the pharmaceuticalcomposition of the present invention will vary, depending on theseverity of the disease being treated and the condition and potentialidiosyncratic response of each individual patient. It is contemplatedthat the duration of each application of the isolated FMH-1 protein willbe in the range of 12 to 24 hours of continuous intravenousadministration. Ultimately the attending physician will decide on theappropriate duration of intravenous therapy using the pharmaceuticalcomposition of the present invention.

[0053] Isolated FMH-1 protein of the invention may also be used toimmunize animals to obtain polyclonal and monoclonal antibodies whichspecifically react with the FMH-1 protein and which may inhibit ligandbinding to the FMH-1. Such antibodies may be obtained using the entireFMH-l protein as an immunogen, or by using fragments of FMH-1 proteinsuch as the soluble mature FMH-1 protein. Smaller fragments of the FMH-1protein may also be used to immunize animals. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus,and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Additional peptide immunogens may be generated by replacing tyrosineresidues with sulfated tyrosine residues. Methods for synthesizing suchpeptides are known in the art, for example, as in R. P. Merrifield,J.Amer.Chem.Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBSLett. 211, 10 (1987).

[0054] Neutralizing or non-neutralizing antibodies (preferablymonoclonal antibodies) binding to FMH-1 protein may also be usefultherapeutics in the treatment of conditions described above. Theseneutralizing monoclonal antibodies are capable of blocking the ligandbinding to the FMH-1 protein.

EXAMPLE 1 Isolation of FMH-1 cDNA

[0055] A partial clone for FMH-2 was isolated from a cDNA library madefrom RNA isolated from stimulated human peripheral blood mononuclearcells using methods as described in Maniatis, et al. The sequence fromthis clone was found to be homologous to another partial clone (herecalled FMH-1) from the EST/IMAGE consortium (T96912). 5′ RACE wasperformed on a cDNA library (Clontech) from unstimulated humanperipheral blood mononuclear cells using the Advantage PCR cDNA kit(Clontech) to obtain a full length clone for FMH-1. Comparison of thissequence to the sequence of the original partial IMAGE clone confirmedidentity and that the isolated cDNA was full length.

[0056] The FMH-1 cDNA was deposited with ATCC on Jun. 26, 1996 ataccession number 98086.

[0057] The sequence of FMH-1 shows regions of homology to FADD(Fas-associating protein with death domain) and to ICE (Interleukin-1beta Converting Enzyme)-like enzymes. This suggests this protein isinvolved in linking Fas and TNF receptor adaptor proteins to theproteases in the signaling pathway leading to apoptosis (programmed celldeath) (see, for example, Nicholson, Nature Biotechnology 14:297(1996)).

[0058] All patent and literature references cited herein areincorporated by reference as if fully set forth.

1 2 1704 base pairs nucleic acid double linear cDNA CDS 121..1683 1CCCGGGCAGG TCTTGGAGCA CACAGAGGAT TCTACTTTCT TTAAAACTTT GTTTTCAGGC 60AATTTCCCTG AGAACCGTTT ACTTCCAGAA GATTGGTGGA GCTTGATCTG AAGGCTGGC 120ATGAAATCTC AAGGTCAACA TTGGTATTCC AGTTCAGATA AAAACTGTAA AGTGAGCTT 180CGTGAGAAGC TTCTGATTAT TGATTCAAAC CTGGGGGTCC AAGATGTGGA GAACCTCAA 240TTTCTCTGCA TAGGATTGGT CCCCAACAAG AAGCTGGAGA AGTCCAGCTC AGCCTCGGA 300GTTTTTGAAC ATCTCTTGGC AGAGGATCTG CTGAGTGAGG AAGACCCTTT CTTCCTGGC 360GAACTCCTCT ATATCATACG GCAGAAGAAG CTGCTGCAGC ACCTCAACTG TACCAAAGA 420GAAGTGGAGC GACTGCTGCC CACCCGACAA AGGGTTTCTC TGTTTAGAAA CCTGCTCTA 480GAACTGTCAG AAGGCATTGA CTCAGAGAAC TTAAAGGACA TGATCTTCCT TCTGAAAGA 540TCGCTTCCCA AAACTGAAAT GACCTCCCTA AGTTTCCTGG CATTTCTAGA GAAACAAGG 600AAAATAGATG AAGATAATCT GACATGCCTG GAGGACCTCT GCAAAACAGT TGTACCTAA 660CTTTTGAGAA ACATAGAGAA ATACAAAAGA GAGAAAGCTA TCCAGATAGT GACACCTCC 720GTAGACAAGG AAGCCGAGTC GTATCAAGGA GAGGAAGAAC TAGTTTCCCA AACAGATGT 780AAGACATTCT TGGAAGCCTT ACCGCAGGAG TCCTGGCAAA ATAAGCATGC AGGTAGTAA 840GGTAACAGAG CCACAAATGG TGCACCAAGC CTGGTCTCCA GGGGGATGCA AGGAGCATC 900GCTAACACTC TAAACTCTGA AACCAGCACA AAGAGGGCAG CTGTGTACAG GATGAATCG 960AACCACAGAG GCCTCTGTGT CATTGTCAAC AACCACAGCT TTACCTCCCT GAAGGACA 1020CAAGGAACCC ATAAAGATGC TGAGATCCTG AGTCATGTGT TCCAGTGGCT TGGGTTCA 1080GTGCATATAC ACAATAATGT GACGAAAGTG GAAATGGAGA TGGTCCTGCA GAAGCAGA 1140TGCAATCCAG CCCATGCCGA CGGGGACTGC TTCGTGTTCT GTATTCTGAC CCATGGGA 1200TTTGGAGCTG TCTACTCTTC GGATGAGGCC CTCATTCCCA TTCGGGAGAT CATGTCTC 1260TTCACAGCCC TGCAGTGCCC TAGACTGGCT GAAAAACCTA AACTCTTTTT CATCCAGG 1320TGCCAAGGTG AAGAGATACA GCCTTCCGTA TCCATCGAAG CAGATGCTCT GAACCCTG 1380CAGGCACCCA CTTCCCTGCA GGACAGTATT CCTGCCGAGG CTGACTTCCT ACTTGGTC 1440GCCACTGTCC CAGGCTATGT ATCCTTTCGG CATGTGGAGG AAGGCAGCTG GTATATTC 1500TCTCTGTGTA ATCATCTGAA GAAATTGGTC CCAAGGATGC TGAAATTTCT GGAAAAGA 1560ATGGAAATCA GGGGCAGGAA GAGAACAGTG TGGGGTGCTA AACAGATCTC AGCAACCT 1620CTGCCCACGG CCATCTCTGC GCAGACACCT CGACCCCCCA TGCGCAGGTG GAGCAGCG 1680TCCTAGTTCT TTCCAGAGGC TTCC 1704 521 amino acids amino acid <Unknown>linear protein 2 Met Lys Ser Gln Gly Gln His Trp Tyr Ser Ser Ser Asp LysAsn Cy 1 5 10 15 Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Ile Asp Ser AsnLeu Gl 20 25 30 Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly LeuVal Pr 35 40 45 Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Ser Asp Val PheGlu Hi 50 55 60 Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe PheLeu Al 65 70 75 80 Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Leu Leu GlnHis Leu As 85 90 95 Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pro Thr ArgGln Arg Va 100 105 110 Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser GluGly Ile Asp Se 115 120 125 Glu Asn Leu Lys Asp Met Ile Phe Leu Leu LysAsp Ser Leu Pro Ly 130 135 140 Thr Glu Met Thr Ser Leu Ser Phe Leu AlaPhe Leu Glu Lys Gln Gl 145 150 155 160 Lys Ile Asp Glu Asp Asn Leu ThrCys Leu Glu Asp Leu Cys Lys Th 165 170 175 Val Val Pro Lys Leu Leu ArgAsn Ile Glu Lys Tyr Lys Arg Glu Ly 180 185 190 Ala Ile Gln Ile Val ThrPro Pro Val Asp Lys Glu Ala Glu Ser Ty 195 200 205 Gln Gly Glu Glu GluLeu Val Ser Gln Thr Asp Val Lys Thr Phe Le 210 215 220 Glu Ala Leu ProGln Glu Ser Trp Gln Asn Lys His Ala Gly Ser As 225 230 235 240 Gly AsnArg Ala Thr Asn Gly Ala Pro Ser Leu Val Ser Arg Gly Me 245 250 255 GlnGly Ala Ser Ala Asn Thr Leu Asn Ser Glu Thr Ser Thr Lys Ar 260 265 270Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg Gly Leu Cys Val Il 275 280285 Val Asn Asn His Ser Phe Thr Ser Leu Lys Asp Arg Gln Gly Thr Hi 290295 300 Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln Trp Leu Gly Phe Th305 310 315 320 Val His Ile His Asn Asn Val Thr Lys Val Glu Met Glu MetVal Le 325 330 335 Gln Lys Gln Lys Cys Asn Pro Ala His Ala Asp Gly AspCys Phe Va 340 345 350 Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala ValTyr Ser Ser As 355 360 365 Glu Ala Leu Ile Pro Ile Arg Glu Ile Met SerHis Phe Thr Ala Le 370 375 380 Gln Cys Pro Arg Leu Ala Glu Lys Pro LysLeu Phe Phe Ile Gln Al 385 390 395 400 Cys Gln Gly Glu Glu Ile Gln ProSer Val Ser Ile Glu Ala Asp Al 405 410 415 Leu Asn Pro Glu Gln Ala ProThr Ser Leu Gln Asp Ser Ile Pro Al 420 425 430 Glu Ala Asp Phe Leu LeuGly Leu Ala Thr Val Pro Gly Tyr Val Se 435 440 445 Phe Arg His Val GluGlu Gly Ser Trp Tyr Ile Gln Ser Leu Cys As 450 455 460 His Leu Lys LysLeu Val Pro Arg Met Leu Lys Phe Leu Glu Lys Th 465 470 475 480 Met GluIle Arg Gly Arg Lys Arg Thr Val Trp Gly Ala Lys Gln Il 485 490 495 SerAla Thr Ser Leu Pro Thr Ala Ile Ser Ala Gln Thr Pro Arg Pr 500 505 510Pro Met Arg Arg Trp Ser Ser Val Ser 515 520

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence comprising a fragment of the nucleotide sequence ofSEQ ID NO: 1 which encodes a protein comprising an amino acid sequencecomprising amino acids 20 to 95 of SEQ ID NO: 2 having FMH-1 proteaseactivity.
 2. The polynucleotide of claim 1 wherein said nucleotidesequence is operably linked to an expression control sequence.
 3. Thepolynucleotide of claim 1 which encodes a human FMH-1 protein.
 4. Thepolynucleotide of claim 1 comprising the nucleotide sequence of SEQ IDNO: 1 from nucleotide 121 to nucleotide
 1683. 5. A host cell transformedwith the polynucleotide of claim
 2. 6. The host cell of claim 5, whereinsaid cell is a mammalian cell.
 7. A process for producing an FMH-1protein, said process comprising: (a) growing a culture of the host cellof claim 5 in a suitable culture medium; and (b) purifying the FMH-1protein from the culture.
 8. The polynucleotide of claim 1 comprising anucleotide sequence comprising a fragment which encodes an amino acidsequence comprising amino acids 20 to 95 of SEQ ID NO: 2.